Membrane traffic researchers have an increasingly detailed view of the mechanisms that drive vesicular transport, but we have only a limited grasp of the events that create, maintain, and transform membrane compartments. Based partly on work from my group, budding yeasts are a powerful system for tackling these questions. We will employ the yeasts Saccharomyces cerevisiae and Pichia pastoris to explore fundamental aspects of membrane compartmentation and organization. Specific Aim #1 is to characterize how cisternal maturation drives secretory cargo transport and Golgi compartmentation using S. cerevisiae. The nonstacked Golgi in S. cerevisiae enables the maturation of individual cisternae to be visualized by fluorescence microscopy. We will use this system to test the assumption that secretory cargo proteins travel through the Golgi in maturing cisternae, and to address the long-standing question of how Golgi cisternae become functionally specialized. Sub-Aim #1A is to track a fluorescent secretory cargo through the Golgi during cisternal maturation. Our hypothesis is that secretory cargo proteins remain in maturing cisternae as resident Golgi proteins come and go. We will test this hypothesis using a novel regulatable fluorescent secretory cargo that can be trapped in the yeast ER and then released for transport through the Golgi. Sub-Aim #1B is to define Golgi organization by a kinetic analysis of resident Golgi proteins. Our hypothesis is that Golgi maturation occurs in discrete kinetic stages that generate functionally distinct types of cisternae. To test this idea and to classify Golgi cisternae, we will perform a systematic, quantitative study of the relative arrival and departure times of resident S. cerevisiae Golgi proteins in wild-type and mutant strains. Specific Aim #2 is to characterize the physical and functional interactions of ER exit sites (ERES) with other compartments using P. pastoris. A typical P. pastoris cell has 3-4 ERES, each of which is next to a Golgi stack. We will explore the relationship of the ERES with the early Golgi and with the pre-autophagosomal structure (PAS). Sub-Aim #2A is to test whether ERES formation requires association with the early Golgi. Our hypothesis is that the unit of self-organization in the early secretory pathway consists of an ERES plus associated early Golgi membranes. To test this idea, we will seek to disrupt the tethers that link the ERES to the early Golgi in P. pastoris, and will determine whether ERES organization is lost in the absence of tethering. Sub-Aim #2B is to test whether PAS assembly occurs in proximity to functionally specialized ERES. Our hypothesis is that specialized ERES in P. pastoris associate with both the vacuole and the PAS. We will take advantage of the simple morphology in this yeast to clarify how the location and dynamics of the PAS relate to those of ERES.